Improving the water solubility of cannabidiol using a peptide carrier

Cannabidiol (CBD), nonpsychotropic cannabinoid found in Cannabis sativa, is a very promising drug candidate offering many differential effects such as sedative, antiinflammatory, antioxidant, and neuroprotective properties. Nevertheless, the therapeutic use of CBD is hindered by its lack of water solubility and relatively low bioavailability. Various carriers have been used to address the solubility issues of CBD and other highly lipophilic drugs so far. However, self-assembled peptide nanostructures as carrier have not been used to improve the water solubility of CBD yet. In this study, a self-assembling peptide micelle was demonstrated to be an effective vehicle for encapsulation of CBD and increased its aqueous solubility up to 2000-fold compared to CBD itself.

reported that the water solubility of CBD in CBD/β-CD IC and CBD/DM-β-CD IC was significantly increased to 0.395 and 14.118 μg/mL, which was enhanced by 17-fold and 614-fold respectively, and the in vitro dissolution rate of CBD was also promoted after complexation [11].
There are also many other studies that used solid lipid nanoparticles (SLNs), polymer micelles, nanoparticles and hybrid nanosystems to encapsulate CBD for delivery purposes [5].None of them directly reported on certain solubility increment of CBD.However, all studies indicate that an increased CBD release is seen due to increased CBD solubility and improved dissolution properties.
Among above-mentioned carrier systems, self-assembling peptides are playing a more and more important role as a carrier due to their inherent biodegradability, biocompatibility, and ability to self-assembling into various nanostructures [14,15].Additionally, their high sensitivity to the microenvironment makes them ideal tools for drug delivery [16].Similar to other examples of aqueous self-assembly, peptide self-assembly is typically built around by an amphiphilic character in monomer units.Amphiphilic peptides having both hydrophilic and hydrophobic domains spontaneously arrange into structures shielding hydrophobic groups by minimizing contact with water.Amphipathicity combined with the functional group richness and modifiability of the peptides enable stable coassembly of some drugs with peptides through strong intermolecular interactions.Due to the aggregation of hydrophobic domains of peptides during selfassembly, these materials may promote drug solubility.Drugs capable of π-π interactions with other aromatic moieties may help to stabilize the assembly [17].Therefore, successful encapsulation and delivery of some hydrophobic drugs using self-assembled peptide nanostructures have been reported [18][19][20].However, so far there is no reported research on using peptide nanostructures for encapsulation CBD.The aim of the current study was to develop peptide-based formulation of CBD that provide higher dispersion than pure CBD in aqueous systems.Considering aromatic content of both CBD and anthracene units, self-assembling APK peptide (Antharecene-Ahx-Pro-Pro-Pro-Lys-Lys-Lys-NH 2 , Ahx: 6-Aminohexanoic acid) has been utilized as a carrier (Figure 1).This newly developed CBD-peptide formulation was characterized with TEM, DLS and FTIR analysis.Increment of solubility of CBD in water was evaluated with HPLC.

Mass spectrometry
High resolution mass spectra (HRMS) of the peptide was recorded on Agilent Technologies 6530 Accurate-Mass Q-TOF.Concentration of the peptide for MS measurement was arranged 0.1 mg/mL with 0.1% formic acid containing ddH 2 O and sample introduction was performed via direct infusion.

Fourier-transform infra-red (FTIR)
Using an ATR-FTIR spectrometer (Nicolet iS10, Thermo Scientific, MA, USA), FTIR analysis was performed on commercial CBD and freeze-dried peptide assemblies with/without CBD.A broad scan of the samples was performed from 600 cm −1 to 4000 cm −1 with a resolution of 0.482 cm −1 .A total of 32 spectra were averaged to reduce noise.The spectra were recorded in transmittance mode at room temperature.The process of the spectral data was completed with the commercially available software OMNIC Series Software (Thermo Fisher Scientific, MA, USA).

Encapsulation of CBD
APK peptide was dissolved in 100 µL HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol) at varying concentrations below their critical gelation concentrations and over their critical aggregation concentrations.Five µL of CBD solution from 16 mM stock solution was pipetted into each peptide solutions and mixtures were sonicated for 30 min at room temperature.Then, HFIP was left evaporation in an open air for 1h at RT.After the removal of HFIP, peptide amphiphiles were reconstituted in water at pH:4 for 20 min at room temperature.The solutions were centrifuged at 12,000 rpm at 20 °C for 30 min to remove any nonencapsulated CBD.The supernatants were analyzed using analytical HPLC (Dionex UltiMate 3000 HPLC system, USA) equipped with a Gemini-NX C18 column-dimensions 150 × 3 mm, particle size 3 μm, pore size 110 Å (Phenomenex, Torrance, California, USA).Peptides were eluted in acetonitrile containing 0.08% TFA (v/v) and water containing 0.1% TFA (v/v) gradient (5%-100%, 1-30 min., flow 0.4 mL/min).The wavelength detected was 214 nm and the volume of each injection was 10 μL.The concentration of solubilized CBD (mM) in aqueous medium surrounded by a peptide micelle was calculated using a standard CBD curve prepared in advance.Encapsulation efficiency (EE%) of CBD in micelles structure were calculated using the following equations: EE% = Amount of CBD in micelles Total amount of CBD added

Dynamic light scattering (DLS) measurements
DLS measurements were carried out on a Zetasizer Nano-ZS equipment (Malvern Instruments, USA).Number average hydrodynamic sizes were obtained by cumulative analysis of autocorrelation data.Samples were placed in polystyrene cells, which were cleaned with ultrapure water.Measurements were taken 5 times at 25 °C in order to check their reproducibility.As previously described in the literature, 5 mM peptide solution was prepared in water and pH of the solution was fixed to pH 4 using 1 M HCl solution to obtain peptide micelles [21].Encapsulation of CBD into micelles were performed using aforementioned solvent coevaporation method.

Transmission electron microscopy (TEM)
Self-assembling peptide micelles impregnated with/without CBD were prepared and then diluted 500 times with water.Three µL of solution was dropped to carbon coated copper grid and then excess sample was removed with a blotting paper after 60 s incubation.Sample coated grid was then wash with 3 µL ddH20.Immediately after removing excess water from grid, 3 µL uranyl acetate (2 wt % ) was pipetted onto grid as negative stain and waited 60 s.Excess stain was removed with a blotting paper.Finally, carbon grids were air-dried at RT for at least 3 h before imaging.Light-field TEM imaging was performed on a FEI Tecnai G2 F30 electron microscope operating at an acceleration voltage of 300 kV.

CBD encapsulation efficiency of peptide micelles
To encapsulate CBD into the peptide micelles, a coassembly method was used to yield quantitative levels of encapsulation [19].In this method, both APK peptide and CBD were mixed in mutually dissolving solvent, which was hexafluoroisopropanol (HFIP).HFIP is a low boiling point solvent and is easily removed by evaporation to produce a film.After the removal of HFIP, the mixture was reconstituted in water.pH of water was adjusted to 4 using few drops of 1 M HCl solution.The amount of CBD loaded was determined by HPLC after removal of nonencapsulated CBD via centrifugation.We found that APK peptide increased a satisfactory CBD solubilization and encapsulation efficiency (Figure 2) .APK peptides are self-assembled into shell-core nanomicelles formed by the hydrophilic (-Lys-Lys-Lys-) and hydrophobic (Anthracene) molecules.Hydrophobic CBD molecules can reside on the core of the shell via π-π stacking and Van der Waals interactions while hydrophilic lysine tails interact with water allowing nanomicelles to solubilize CBD in aqueous medium.CBD shows a low water solubility, measured at 0.1 µg/mL (3.18 × 10 -4 mM) [22,23].When using the micelle forming APK peptide to encapsulate CBD, aqueous solubility values as high as 0.64 mM CBD were observed, which is more than 2000-fold higher than the solubility reported for CBD alone in water.When encapsulating a 0.795 mM initial concentration of CBD, an efficiency of 80.4% was observed for a 4 mM APK solution.A further increase in peptide concentration did not result in any further increase in the solubility of CBD, but solubility of CBD remained almost same against increased peptide concentration.To the best of our knowledge, 80.4% EE and 2000-fold increase in solubility for CBD are the highest values ever reported using any type of nanocarrier systems (polymer, peptide or inorganic nanoparticles) [24].

Structural analysis of CBD loaded peptide carriers
After encapsulation of CBD into peptides using coassembly method, nonencapsulated CBD was removed with high-speed centrifugation and resulting aqueous mixture was freeze-dried.Beige color solid was analyzed with FT-IR and HPLC to confirm the residence of CBD into the carrier.
FT-IR method was employed for the evaluation of the chemical composition of the micelles after CBD incorporation and the possible interactions between CBD and amino acid side chain of peptides.FTIR spectrum of CBD loaded peptide carrier possessed the characteristic peaks of CBD, confirming the successful loading of CBD.CBD has two distinct peaks observed at 1579 cm −1 and 1624 cm −1 , possibly due to the C=C stretching vibration of the compound and has a characteristic absorption band at 3515 and 3407 cm −1 , possibly due to the vibration of the phenolic -OH groups [25].Additionally, peak at 1377 cm −1 assigned for the bending vibration of methyl groups, and peak at 1215 cm -1 denoted for the C-O stretching vibration in CBD molecule [11].If CBD incorporated into the micelles, some of high-intensity peaks of pure CBD is expected to shift or disappear due to restriction of the above-mentioned groups inside the micelle core.As seen in Figure 3, the characteristic peaks of CBD at 3515 cm -1 , 3407 cm -1 , 1624 cm -1 , 1579 cm -1 , 1377 cm -1 and 1215 cm -1 were all disappeared after CBD incorporation into the micelles.It was worth noting that the spectra of APK and APK-CBD micelles were similar to those of APK because of the low content of CBD in the carrier system.Therefore, FTIR spectra of the physical mixture of APK and CBD were also analyzed to show that there is no interaction between CBD and peptide when they were mixed physically.The findings showed that CBD had entirely or partly entered the cavity of the peptide micelles, resulting in that the vibration of the above-mentioned groups was restricted.
Encapsulation of the CBD into micelles further endorsed with HPLC analysis.After encapsulation process, carrier peptides that were expected to contain CBD analyzed with HPLC and presence of CBD was confirmed (Figure 4).

DLS analysis of micelles
DLS measurements of CBD-loaded micelles were carried out to determine the size distribution of micelles with/without CBD.The intensity-weighted plot revealed a hydrodynamic diameter (Dh) distribution of micelles over a fairly wide range in the range of about 100-1200 nm without CBD molecules (Figure 5A).Average hydrodynamic size of the micelles after CBD incubation were also measured to determine if incorporation of CBD induces swelling or shrinkage of the nanostructure.Similarly, the formation of micelles of different sizes (300-5000 nm) has been observed after CBD encapsulation (Figure 5B).Although not significant, incorporation of hydrophobic CBD into core of micelles which was mainly formed stacked anthracene moieties seems to cause some expansion of the micelle size.Presence of CBD in the core of the micelle seems to cause hindrance for stacking of anthracene molecules and cause some shift of sizes.

TEM analysis of micelles
After and before encapsulating CBD into the peptide carriers, conventional TEM was used to visualize their individual morphology.TEM images proved that peptides self-assembled to form micellar structure both presence and absence of CBD (Figure 6).Any consistency about size of micelles was not observed.The presence of many large and small micelles was detected in the TEM images.The overall increase in diameter of peptide with CBD compared to peptide alone showed once again, CBD disturbed the stacked anthracene molecules in core.Both DLS and TEM analysis suggested that a small degree of swelling of the micelles might occur in order to accommodate a hydrophobic cargo.

Conclusion
This work showed that solubility of CBD can be improved up to 2000-fold after inclusion of CBD in self-assembled peptide micelles.CBD was trapped into the peptide micelles using solvent coevaporation method.The presence of CBD in peptide nanostructures were confirmed both in FTIR spectra analysis and HPLC peak assignment.Encapsulation efficiency of peptide micelles and solubility of CBD embedded in micelles were calculated after removal of nonencapsulated CBD from the carrier system.Morphological analysis of peptide micelles with TEM showed that overall micelle size was expanding after the encapsulation.This could be explained as CBD residing in the hydrophobic core is causing sort of disruption of anthracene moieties that forms the interior core.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figure 1 .
Figure 1.Sequence of APK peptide and structure of CBD molecule.CBD was encapsulated into a micellar peptide carrier via coassembly.

Figure 2 .
Figure 2. Peptide amphiphile nanomicelle encapsulation of cannabidiol.The initial concentration of CBD was kept constant at 0.795 mM while the concentration of peptide was gradually increased up to 4mM.